A thrombus is the result of processes which initiate the coagulation cascade. It is composed of an aggregation of platelets enmeshed in a polymeric network of fibrin. This process is normally initiated as a consequence of tissue injury and has the effect of slowing or preventing blood flow in a vessel. Etiological factors which are not directly related to tissue injury, such as atherosclerotic plaque, inflammation of the blood vessels (phlebitis) and septicemia, may also initiate thrombus formation. In some instances, the inappropriate formation of a thrombus, and subsequent decrease in blood flow, may have pathological consequences, such as stroke, pulmonary embolism and heart disease.
Platelets play a major role in thrombus formation. Current antithrombotic therapy employs agents that modify the platelet/endothelial cell arachidonate-prostaglandin system, such as prostacyclin analogues, cyclooxygenase inhibitors, thromboxane synthesis, inhibitors and thromboxane receptor antagonists; and anti-coagulants, such as heparin. These agents inhibit one or both of two discernible phases of platelet aggregation. The primary phase, which is a response to chemical stimuli, such as ADP (adenosine diphosphate), collagen, epinephrine or thrombin, causes initial activation of the platelets. This is followed by a secondary phase, which is initiated by the platelets themselves, and is characterized by thromboxane A.sub.2 (TxA.sub.2) synthesis and the release of additional ADP from platelet storage granules, which further activates platelets.
Prostacyclin, also called prostaglandin I.sub.2 (PGI.sub.2), and stable PGI.sub.2 analogues inhibit both the primary and secondary phases of platelet aggregation. However, use of such analogues has been associated with undesirable changes in blood pressure. See Aiken, et al., Prostaglandins, 19, 629-43 (1980).
Cyclooxygenase inhibitors and thromboxane synthetase inhibitors act to block the production of TxA.sub.2. TxA.sub.2 antagonists block the effects of TxA.sub.2 by binding the TxA.sub.2 receptor. These therapies act only upon the secondary stage of platelet activation. Use of cyclooxygenase inhibitors has been associated with ulcerogenesis and an adverse effect upon prostacyclin synthesis.
Heparin prevents the activation of fibrinogen by thrombin and thereby prevents the activation of the GPIIb-IIIa receptor by thrombin. This inhibits only the primary phase of platelet aggregation and has little effect upon activation of platelets by other means, such as collagen, ADP and epinephrine.
Cyclooxygenase inhibitors, prostaglandin analogues and heparin all inhibit platelet aggregation indirectly by inhibiting the primary or secondary phase of platelet/fibrinogen activation. There is therefore a need for selective therapeutic products which block platelet aggregation directly, whether it arises from the primary or secondary phase of platelet activation.
Platelet aggregation is believed to be mediated primarily through the GPIIb-IIIa platelet receptor complex. Von Willebrand factor, a plasma protein, and fibrinogen are able to bind and crosslink GPIIb-IIIa receptors on adjacent platelets and thereby effect aggregation of platelets. Fibronectin, vitronectin and thrombospondin are proteins which have also been demonstrated to bind to GPIIb-IIIa. Fibronectin is found in plasma and as a structural protein in the intracellular matrix. Binding between the structural proteins and GPIIb-IIIa may function to cause platelets to adhere to damaged vessel walls.
The importance of the GPIIb-IIIa receptor to platelet aggregation has been demonstrated by methods which mask the receptor. Thus, Coller et al, (Blood, 66, 1456-9 (1985)) have shown that antibodies to this complex inhibit platelet aggregation in dogs induced by ADP.
Peptide fragments of human plasma fibronectin and synthetic peptides containing the RGD sequence which promote cell attachment and enhance phagocytosis are disclosed in U.S. Pat. Nos. 517,686, 4,589,881, 4,661,111 and 4,614,517. Linear and cyclic peptides containing an RGD sequence have also be reported in WO 89/05150 (PCT US88/04403). Peptides which contain an RGD sequence have been reported to inhibit platelet aggregation. Nievelstein et al, (Thromb. and Hemostasis, 58, 2133(1987)) have reported that -RGDS- peptides inhibit thrombin induced aggregation and adhesion of platelets to fibronectin, and may interact through the GPIIb-IIIa complex. U.S. Pat. No. 4,683,291 discloses peptides containing Arg and Lys and an -RGD- sequence which inhibit binding of fibrinogen to platelets and inhibit platelet aggregation. A disadvantage of these peptides is their poor stability in plasma and their low potency. EP 0 275 748 discloses linear tetra- to hexapeptides and cyclic hexa- to octapeptides which bind to the GPIIb-IIIa receptor and inhibit platelet aggregation. The cyclic peptides reported are formed via a disulfide bridge between two cysteinyl residues. Other linear and cyclic peptides, the disclosure of which are incorporated herein by reference, are reported in EP-A 0 341 915. These cyclic structures comprise a disulfide ring formed by two sulfhydryl-bearing aliphatic amino acid residues.
The instant invention provides cyclic compounds in which the cyclic structure comprises a homodetic peptide wherein the ring is formed by a peptide bond, or a heterodetic peptide wherein the ring is formed by an alkylene, sulfide or disulfide bridge. This invention further discloses cyclic compounds in which the unit B-Gly-Asp-Q, as hereinafter defined in formula (I), is repeated more than once. Construction of such unusual ring structures surprisingly results in pharmacologically active compounds. It has further been discovered that neither a terminal amino group or a terminal carbonyl group is required for anti-aggregatory activity. The compounds of this invention are further resistant to plasma proteases and show a selectivity for inhibition of binding to the fibrinogen receptor over other integrin receptors, such as vitronectin or fibronectin. Thus, an advantage to the compounds of this invention is their ability to inhibit platelet aggregation without appreciably inhibiting the adhesion of platelets to other integrin receptors.
Recent advances for treatment of occluded arteries and deep vein thrombosis employ fibrinolytic agents to lyse thrombi or emboli in order to reestablish or improve blood flow. Fibrinolytic agents, such as tissue plasminogen activator (tPA), urokinase (UK), pro-Urokinase(pUK), and streptokinase (SK), and mutants and derivatives thereof, are proteolytic enzymes which cause fibrin to be hydrolyzed at specific sites and thereby fragment the fibrin network. Their action in vivo is to proteolytically activate plasminogen in the blood to form plasmin, which causes lysis of the fibrin clot. Lysis of fibrin into smaller peptides has the effect of solubilizing the thrombus or embolus. A recurrent problem with such therapy, however, is the reocclusion of the blood vessel due to formation of a secondary thrombus.
Fibrinolytic therapy is most commonly used for reestablishing flow in a thrombosed blood vessel. However, fibrinolytic therapy does not reverse the factors responsible for the initiation of the thrombus. For this reason, anticoagulants such as heparin are often used to prevent reocclusion. In fact, patients which have a high degree of stenosis in an artery are at extremely high risk of rethrombosis after reperfusion, even in the presence of high doses of heparin. See Gold et al., Circ., 73, 347-52 (1986). In addition, use of SK and tPA has been associated with platelet hyperaggregability. See Ohlstein, et al., Thromb. Res., 4, 575-85 (1987). Treatment with higher doses of tPA can be associated with systemic bleeding and is not recommended for preventing reocclusion. There is, therefore, a need for a method for preventing rethrombosis after fibrinolytic therapy.
U.S. patent application Ser. No. 917,122 discloses TxA.sub.2 antagonists for use in a method for inhibiting reocclusion following reperfusion and for lowering the dose of tPA required for fibrinolysis. Yasuda et al. (Clin. Res., 34, 2 (1986)) have demonstrated that reocclusion by fibrin rich platelet thrombi, after thrombolysis with tPA, may be inhibited by a murine monoclonal antibody to GPIIb-IIIa in dogs. This invention discloses a new method for inhibiting reocclusion of a blood vessel by administering peptides which directly inhibit platelet aggregation.